Yeast promotors for protein expression

ABSTRACT

Isolated nucleic acids, expression methods, host cells, expression vectors, and DNA constructs for producing proteins, and proteins produced using the expression methods are disclosed. More specifically, nucleic acids isolated from  Pichia  pastons having promoter activity and expression methods, host cells, expression vectors, and DNA constructs of using the  Pichia  pastons promoters to produce different proteins and polypeptides are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. Ser. No.61/775,029, filed Mar. 8, 2013, incorporated herein by reference.

FIELD OF THE DISCLOSURE

The present invention is related to isolated nucleic acids, expressionmethods, host cells, expression vectors, and DNA constructs forproducing proteins, and polypeptides, and to the proteins and thepolypeptides produced using the expression methods. More particularly,the invention relates to nucleic acids isolated from Pichia pastoriswherein the nucleic acids have promoter activity. The invention alsorelates to expression methods, host cells, expression vectors, and DNAconstructs, for using the Pichia pastoris promoters to produce proteinsand polypeptides, and to the proteins and the polypeptides producedusing the expression methods.

BACKGROUND AND SUMMARY OF THE INVENTION

Yeast expression systems can be used to effectively produce proteins,such as enzymes, hormones, and vaccine proteins, in part, because someyeast grow rapidly to high cell densities, are grown in simple andinexpensive media, and are eukaryotes so they can modify proteins in amanner similar to native proteins in mammals. Additionally, with aproper signal sequence, the expressed protein can be secreted into theculture medium for convenient isolation and purification. Some yeastexpression systems are also accepted in the food and pharmaceuticalindustries as being safe for the production of pharmaceuticals and foodproducts, unlike fungal and bacterial expression systems which may insome cases be unsafe, for example, for human food manufacturing.

Thus, it is beneficial for a variety of industries, such as the food andanimal feed industries, the human and animal health industries, and thelike, to develop or improve yeast expression systems that can be used toexpress high levels of proteins to increase yield, reduce the expense ofisolation and purification of proteins, and reduce the costs of humanand animal health products and food products.

A variety of types of yeast expression systems have been developedinvolving either the use of inducible or constitutive expression ofproteins using nucleic acids encoding homologous or heterologousproteins, under the control of a yeast promoter. Promoters areregulatory elements that are linked to the 5′ end of a nucleic acidencoding a protein, and may interact with various regulatory factors inthe host cell (e.g., a yeast host cell) to control transcription of RNAfrom DNA. Promoters may also control the timing of transcription of RNAfrom DNA. For example, the AOX 1 promoter has been identified in theyeast Pichia pastoris, and is commonly used in yeast expression systemsbecause it is a tightly regulated, strong promoter.

Due to the importance of yeast expression systems for a variety ofindustries, including the human pharmaceuticals industry, and the humanfood and animal feed industries, the improvement of yeast expressionsystems is the focus of much research and development. Accordingly, thepresent inventors have identified promoters from Pichia pastoris thatare particularly effective for use in expression of proteins in yeast.The promoters described herein can be used, for example, inmethanol-inducible yeast expression systems, or in expression systemsfor the constitutive expression of proteins.

In one illustrative embodiment of the invention, an isolated nucleicacid is provided wherein the sequence of the isolated nucleic acidcomprises a sequence, for example, at least 90%, 95%, or 98% identicalto a sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 asdescribed herein, or at least 90%, 95%, or 98% identical to a fragmentthereof, wherein the isolated nucleic acid comprises the sequence of amethanol-inducible Pichia pastoris promoter. In other embodiments,expression vectors, host cells, and DNA constructs comprising thesepromoter sequences are provided.

In another embodiment, a method of producing a protein using thesepromoter sequences is provided. The method comprises the steps ofculturing in a culture medium a host cell comprising a first expressioncassette comprising any of the above promoter sequences operably linkedto a heterologous coding sequence encoding a protein, wherein theculturing is done under conditions permitting expression of the protein.In another illustrative embodiment, an isolated protein producedaccording to this method is provided.

In one illustrative embodiment of the invention, an isolated nucleicacid is provided wherein the sequence of the isolated nucleic acidcomprises a sequence, for example, at least 90%, 95%, or 98% identicalto a sequence selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, and SEQ ID NO:14 as described herein, or at least 90%, 95%, or98% identical to a fragment thereof, wherein the isolated nucleic acidcomprises the sequence of a constitutive Pichia pastoris promoter. Inother embodiments, expression vectors, host cells, and DNA constructscomprising these promoter sequences are provided.

In another embodiment, a method of producing a protein using thesepromoter sequences is provided. The method comprises the steps ofculturing in a culture medium a host cell comprising a first expressioncassette comprising any of the above promoter sequences operably linkedto a heterologous coding sequence encoding a protein, wherein theculturing is done under conditions permitting expression of the protein.In another illustrative embodiment, an isolated protein producedaccording to this method is provided.

All of the embodiments described in the following clause list are alsocontemplated for use in accordance with the invention. For all of theembodiments described in the following clauses, any applicablecombination of embodiments is considered to be in accordance with theinvention.

1. An isolated nucleic acid wherein the sequence of the isolated nucleicacid comprises a sequence at least 90% identical to a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 90% identical to afragment thereof, wherein the isolated nucleic acid comprises thesequence of a methanol-inducible Pichia pastoris promoter.

2. The isolated nucleic acid of clause 1 wherein the sequence of theisolated nucleic acid is at least 95% identical to a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 95% identical to afragment thereof.

3. The isolated nucleic acid of clause 1 wherein the sequence of theisolated nucleic acid is at least 98% identical to a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 98% identical to afragment thereof.

4. The isolated nucleic acid sequence of clause 1 wherein the sequenceof the isolated nucleic acid is a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6, or a fragment thereof.

5. The isolated nucleic acid of any one of clauses 1 to 4 operablylinked to a heterologous coding sequence.

6. The isolated nucleic acid of clause 5 wherein the heterologous codingsequence encodes a protein selected from the group consisting of atoxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, astructural protein, an immunogenic protein, and a cell signalingprotein.

7. The isolated nucleic acid of clause 6 wherein the protein is anenzyme for use in animal feed.

8. The isolated nucleic acid of clause 7 wherein the protein is selectedfrom the group consisting of a phytase, a mannanase, a galactosidase, anamylase, a glucanase, a protease, a cellulase, and a xylanase.

9. The isolated nucleic acid of clause 8 wherein the protein is aphytase.

10. The isolated nucleic acid of clause 8 wherein the protein is agalactosidase.

11. An expression vector comprising the isolated nucleic acid of any oneof clauses 1 to 10.

12. A host cell comprising the expression vector of clause 11.

13. A host cell comprising the isolated nucleic acid of any one ofclauses 1 to 10.

14. The host cell of any one of clauses 12 or 13 wherein the host cellis a Pichia species.

15. The host cell of clause 14 wherein the Pichia species is Pichiapastoris.

16. A DNA construct comprising the isolated nucleic acid of any one ofclauses 1 to 10.

17. A method of producing a protein, the method comprising the step of

culturing in a culture medium a host cell comprising a first expressioncassette comprising the isolated nucleic acid of any one of clauses 1 to4 operably linked to a heterologous coding sequence encoding a protein,wherein the culturing is done under conditions permitting expression ofthe protein.

18. The method of clause 17 wherein the protein is selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

19. The method of clause 18 wherein the protein is an enzyme for use inanimal feed.

20. The method of clause 19 wherein the protein is selected from thegroup consisting of a phytase, a mannanase, a galactosidase, an amylase,a glucanase, a cellulase, a protease, and a xylanase.

21. The method of clause 20 wherein the protein is a phytase.

22. The method of clause 20 wherein the protein is a galactosidase.

23. The method of any one of clauses 17 to 22 wherein the protein isexpressed using the first expression cassette in combination with asecond expression cassette.

24. The method of clause 23 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising thesequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ IDNO:16 have promoter activity, or any other AOX 1 or AOX 2 promotersequence.

25. The method of clause 24 wherein the protein is expressed using thefirst expression cassette, the second expression cassette, and a thirdexpression cassette.

26. The method of clause 25 wherein the third expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence at least 90%identical to a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6, or at least 90% identical to a fragment thereof.

27. The method of clause 23 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence at least 90%identical to a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6, or at least 90% identical to a fragment thereof.

28. An isolated protein produced according to the method of any one ofclauses 17 to 27.

29. An isolated nucleic acid wherein the sequence of the isolatednucleic acid comprises a sequence at least 90% identical to a sequenceselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14, or at least 90% identical to a fragment thereof, wherein thenucleic acid comprises the sequence of a constitutive Pichia pastorispromoter.

30. The isolated nucleic acid of clause 29 wherein the sequence of theisolated nucleic acid is at least 95% identical to a sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 orat least 95% identical to a fragment thereof.

31. The isolated nucleic acid of clause 29 wherein the sequence of theisolated nucleic acid is at least 98% identical to a sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO: SEQ ID NO: SEQ ID NO:13, and SEQ ID NO: or at least98% identical to a fragment thereof.

32. The isolated nucleic acid sequence of clause 29 wherein the sequenceof the isolated nucleic acid is a sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or a fragmentthereof.

33. The isolated nucleic acid of any one of clauses 29 to 32 operablylinked to a heterologous coding sequence.

34. The isolated nucleic acid of clause 33 wherein the heterologouscoding sequence encodes a protein selected from the group consisting ofa toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine,a structural protein, an immunogenic protein, and a cell signalingprotein.

35. The isolated nucleic acid of clause 34 wherein the protein is anenzyme for use in animal feed.

36. The isolated nucleic acid of clause 35 wherein the protein isselected from the group consisting of a phytase, a mannanase, agalactosidase, an amylase, a glucanase, a cellulase, a protease, and axylanase.

37. The isolated nucleic acid of clause 36 wherein the protein is aphytase.

38. The isolated nucleic acid of clause 36 wherein the protein is agalactosidase.

39. An expression vector comprising the isolated nucleic acid of any oneof clauses 29 to 38.

40. A host cell comprising the expression vector of clause 39.

41. A host cell comprising the isolated nucleic acid of any one ofclauses 29 to 38.

42. The host cell of any one of clauses 40 or 41 wherein the host cellis a Pichia species.

43. The host cell of clause 42 wherein the Pichia species is Pichiapastoris.

44. A DNA construct comprising the isolated nucleic acid of any one ofclauses 29 to 38.

45. A method of producing a protein, the method comprising the step ofculturing in a culture medium a host cell comprising a first expressioncassette comprising the isolated nucleic acid of any one of clauses 29to 38 operably linked to a heterologous coding sequence encoding aprotein, wherein the culturing is done under conditions permittingexpression of the protein.

46. The method of clause 45 wherein the protein is selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

47. The method of clause 46 wherein the protein is an enzyme for use inanimal feed.

48. The method of clause 47 wherein the protein is selected from thegroup consisting of a phytase, a mannanase, a galactosidase, an amylase,a glucanase, a cellulase, a protease, and a xylanase.

49. The method of clause 48 wherein the protein is a phytase.

50. The method of clause 48 wherein the protein is a galactosidase.

51. The method of any one of clauses 45 to 50 wherein the protein isexpressed using the first expression cassette in combination with asecond expression cassette.

52. The method of clause 51 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising thesequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ IDNO:16 have promoter activity, or any other AOX 1 or AOX 2 promotersequence.

53. The method of clause 51 wherein the protein is expressed using thefirst expression cassette, the second expression cassette, and a thirdexpression cassette.

54. The method of clause 53 wherein the third expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence at least 90%identical to a sequence selected from the group consisting of SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, or at least 90% identical to a fragment thereof.

55. The method of clause 51 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence at least 90%identical to a sequence selected from the group consisting of SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, or at least 90% identical to a fragment thereof.

56. An isolated protein produced according to the method of any one ofclauses 45 to 55.

57. The host cell of any one of clauses 40 or 41 wherein the host cellis selected from the group consisting of Hansenula species, Pichiaspecies, Saccharomyces species, Schizosaccharomyces species, Torulasporaspecies, a Candida species, a Yarrowia species, and Kluveromycesspecies.

58. A host cell comprising the DNA construct of clause 44 wherein thehost cell is selected from the group consisting of Hansenula species,Pichia species, Saccharomyces species, Schizosaccharomyces species,Torulaspora species, a Candida species, a Yarrowia species, andKluveromyces species.

59. The method of any one of clauses 45 to 55 wherein the host cell isselected from the group consisting of Hansenula species, Pichia species,Saccharomyces species, Schizosaccharomyces species, a Yarrowia species,Torulaspora species, Candida species, and Kluveromyces species.

60. The host cell of any one of clauses 12 or 13 wherein the host cellis a methylotrophic yeast.

61. The host cell of clause 60 wherein the host cell is selected fromthe group consisting of Hansenula species, Pichia species, and Candidaspecies.

62. A host cell comprising the DNA construct of clause 16 wherein thehost cell is a methylotrophic yeast.

63. The host cell of clause 62 selected from the group consisting ofHansenula species, Pichia species, and Candida species.

64. The method of any one of clauses 17 to 27 wherein the host cell is amethylotrophic yeast.

65. The method of clause 64 wherein the host cell is selected from thegroup consisting of Hansenula species, Pichia species, and Candidaspecies.

66. The method of any one of clauses 25 or 53 wherein the thirdexpression cassette comprises the heterologous coding sequence encodingthe protein operably linked to an isolated nucleic acid having asequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ IDNO:16 have promoter activity, or any other AOX 1 or AOX 2 promotersequence.

67. A method of producing one or more proteins, the method comprisingthe step of culturing in a culture medium a host cell comprising a firstexpression cassette, a second expression cassette, and one or moreadditional expression cassettes, wherein each of the one or moreadditional expression cassettes comprises the isolated nucleic acid ofany one of clauses 1 to 4 operably linked to a heterologous codingsequence encoding the one or more proteins, wherein the culturing isdone under conditions permitting expression of the one or more proteins.

68. A method of producing one or more proteins, the method comprisingthe step of

culturing in a culture medium a host cell comprising a first expressioncassette, a second expression cassette, and one or more additionalexpression cassettes, wherein each of the one or more additionalexpression cassettes comprises the isolated nucleic acid of any one ofclauses 29 to 38 operably linked to a heterologous coding sequenceencoding the one or more proteins, wherein the culturing is done underconditions permitting expression of the one or more proteins.

69. The method of any one of clauses 17 or 45 further comprising thestep of purifying the protein from the medium of the cultured host cell.

70. The method of any one of clauses 67 or 68 further comprising thestep of purifying one or more of the one or more proteins from themedium of the cultured host cell.

71. The isolated nucleic acid, host cell, expression vector, isolatedprotein, DNA construct, or method of any one of clauses 1 to 70 whereinthe isolated nucleic acid consists of any one of SEQ ID NOS. 1 to 14, ora fragment thereof.

72. An isolated nucleic acid consisting of any one of SEQ ID NOS. 1 to14, or a fragment thereof.

73. The isolated nucleic acid of clause 5 wherein the heterologouscoding sequence encodes a protein or a polypeptide selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

74. The isolated nucleic acid of clause 29 wherein the heterologouscoding sequence encodes a protein or a polypeptide selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

75. A method of producing a protein or a polypeptide, the methodcomprising the step of

culturing in a culture medium a host cell comprising a first expressioncassette comprising the isolated nucleic acid of any one of clauses 1 to4 operably linked to a heterologous coding sequence encoding the proteinor the polypeptide, wherein the culturing is done under conditionspermitting expression of the protein or the polypeptide.

76. A method of producing a protein or a polypeptide, the methodcomprising the step of culturing in a culture medium a host cellcomprising a first expression cassette comprising the isolated nucleicacid of any one of clauses 29 to 38 operably linked to a heterologouscoding sequence encoding the protein or the polypeptide, wherein theculturing is done under conditions permitting expression of the proteinor the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a methanol-inducible promoter,CAM1_(FR839630_178837 . . . 180488)_selection (SEQ ID NO:1).

FIG. 2 shows the nucleotide sequence of a methanol-inducible promoter,PP7435_Chr1-1351_selection (SEQ ID NO:2).

FIG. 3 shows the nucleotide sequence of a methanol-inducible promoter,THI4_selection (SEQ ID NO:3).

FIG. 4 shows the nucleotide sequence of a methanol-inducible promoter,GPM2_selection (SEQ ID NO:4).

FIG. 5 shows the nucleotide sequence of a methanol-inducible promoter,PP7435_Chr2-0790_selection (SEQ ID NO:5).

FIG. 6 shows the nucleotide sequence of a methanol-inducible promoter,PP7435_Chr3-0842_selection (SEQ ID NO:6).

FIG. 7 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr1-0269_selection (SEQ ID NO:7).

FIG. 8 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr2-0207_selection (SEQ ID NO:8).

FIG. 9 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr2-0208_selection (SEQ ID NO:9).

FIG. 10 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr2-0809_selection (SEQ ID NO:10).

FIG. 11 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr4-0069_selection (SEQ ID NO:11).

FIG. 12 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr4-0800_selection (SEQ ID NO:12).

FIG. 13 shows the nucleotide sequence of a constitutive promoter,TEF2_selection Pichia pastoris CBS 7435 chromosome 1, complete repliconsequence (SEQ ID NO:13).

FIG. 14 shows the nucleotide sequence of a constitutive promoter,PP7435_Chr3-0476_selection Pichia pastoris CBS 7435 chromosome 3,complete replicon sequence (SEQ ID NO:14).

FIG. 15. shows the nucleotide sequence of the AOX1 promoter sequence(SEQ ID NO:15).

FIG. 16. shows the genomic sequence 1000 bp upstream of the AOX1promoter ATG start codon (SEQ ID NO:16).

FIG. 17. shows the reporter plasmid for testing expression ofalpha-galactosidase (A-gal) with the promoters.

FIG. 18 (panels a-c). shows the images of A-gal expression from Pichiaclones with the reporter plasmid and different promoters.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

In one illustrative embodiment of the invention, an isolated nucleicacid is provided wherein the sequence of the isolated nucleic acidcomprises a sequence, for example, at least 90%, 95%, or 98% identicalto a sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 asdescribed herein, or at least 90%, 95%, or 98% identical to a fragmentthereof, wherein the nucleic acid comprises the sequence of amethanol-inducible Pichia pastoris promoter. In another embodiment, theisolated nucleic acid sequence is a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6, or a fragment thereof. In other embodiments,expression vectors, host cells, and DNA constructs comprising thesepromoter sequences are provided.

In another embodiment, a method of producing a protein using thesepromoter sequences is provided. The method comprises the steps ofculturing in a culture medium a host cell comprising a first expressioncassette comprising any of the above promoter sequences operably linkedto a heterologous coding sequence encoding a protein, wherein theculturing is done under conditions permitting expression of the protein.In another illustrative embodiment, an isolated protein producedaccording to this method is provided.

In one illustrative embodiment of the invention, an isolated nucleicacid is provided wherein the sequence of the isolated nucleic acidcomprises a sequence, for example, at least 90%, 95%, or 98% identicalto a sequence selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, and SEQ ID NO:14 as described herein, or at least 90%, 95%, or98% identical to a fragment thereof, wherein the nucleic acid comprisesthe sequence of a constitutive Pichia pastoris promoter. In anotherembodiment, the isolated nucleic acid sequence is a sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, ora fragment thereof. In other embodiments, expression vectors, hostcells, and DNA constructs comprising these promoter sequences areprovided. The above-described promoters have been isolated from a yeaststrain (i.e., NRRL Y11430) currently classified as a Pichia pastorisyeast strain. However, the classification may change at some point to aKomagataella species (e.g., Komagataella phaffii).

In another embodiment, a method of producing a protein using any of thepromoter sequences in the preceding paragraph is provided. The methodcomprises the steps of culturing in a culture medium a host cellcomprising a first expression cassette comprising any of the abovepromoter sequences operably linked to a heterologous coding sequenceencoding a protein, wherein the culturing is done under conditionspermitting expression of the protein. In another illustrativeembodiment, an isolated protein produced according to this method isprovided.

All of the embodiments described in the following clause list arecontemplated for use in accordance with the invention. For all of theembodiments described in the following clauses, any applicablecombination of embodiments is considered to be in accordance with theinvention. Any embodiment described in the following clause list is alsocontemplated for use with any embodiment described in the Summary ofInvention section of this application or in the Detailed Description ofthe Illustrative Embodiments section of this application.

1. An isolated nucleic acid wherein the sequence of the isolated nucleicacid comprises a sequence at least 90% identical to a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 90% identical to afragment thereof, wherein the isolated nucleic acid comprises thesequence of a methanol-inducible Pichia pastoris promoter.

2. The isolated nucleic acid of clause 1 wherein the sequence of theisolated nucleic acid is at least 95% identical to a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 95% identical to afragment thereof.

3. The isolated nucleic acid of clause 1 wherein the sequence of theisolated nucleic acid is at least 98% identical to a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or at least 98% identical to afragment thereof.

4. The isolated nucleic acid sequence of clause 1 wherein the sequenceof the isolated nucleic acid is a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6, or a fragment thereof.

5. The isolated nucleic acid of any one of clauses 1 to 4 operablylinked to a heterologous coding sequence.

6. The isolated nucleic acid of clause 5 wherein the heterologous codingsequence encodes a protein selected from the group consisting of atoxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, astructural protein, an immunogenic protein, and a cell signalingprotein.

7. The isolated nucleic acid of clause 6 wherein the protein is anenzyme for use in animal feed.

8. The isolated nucleic acid of clause 7 wherein the protein is selectedfrom the group consisting of a phytase, a mannanase, a galactosidase, anamylase, a glucanase, a protease, a cellulase, and a xylanase.

9. The isolated nucleic acid of clause 8 wherein the protein is aphytase.

10. The isolated nucleic acid of clause 8 wherein the protein is agalactosidase.

11. An expression vector comprising the isolated nucleic acid of any oneof clauses 1 to 10.

12. A host cell comprising the expression vector of clause 11.

13. A host cell comprising the isolated nucleic acid of any one ofclauses 1 to 10.

14. The host cell of any one of clauses 12 or 13 wherein the host cellis a Pichia species.

15. The host cell of clause 14 wherein the Pichia species is Pichiapastoris.

16. A DNA construct comprising the isolated nucleic acid of any one ofclauses 1 to 10.

17. A method of producing a protein, the method comprising the step of

culturing in a culture medium a host cell comprising a first expressioncassette comprising the isolated nucleic acid of any one of clauses 1 to4 operably linked to a heterologous coding sequence encoding a protein,wherein the culturing is done under conditions permitting expression ofthe protein.

18. The method of clause 17 wherein the protein is selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

19. The method of clause 18 wherein the protein is an enzyme for use inanimal feed.

20. The method of clause 19 wherein the protein is selected from thegroup consisting of a phytase, a mannanase, a galactosidase, an amylase,a glucanase, a cellulase, a protease, and a xylanase.

21. The method of clause 20 wherein the protein is a phytase.

22. The method of clause 20 wherein the protein is a galactosidase.

23. The method of any one of clauses 17 to 22 wherein the protein isexpressed using the first expression cassette in combination with asecond expression cassette.

24. The method of clause 23 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising thesequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ IDNO:16 have promoter activity, or any other AOX 1 or AOX 2 promotersequence.

25. The method of clause 24 wherein the protein is expressed using thefirst expression cassette, the second expression cassette, and a thirdexpression cassette.

26. The method of clause 25 wherein the third expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence at least 90%identical to a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6, or at least 90% identical to a fragment thereof.

27. The method of clause 23 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence at least 90%identical to a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6, or at least 90% identical to a fragment thereof.

28. An isolated protein produced according to the method of any one ofclauses 17 to 27.

29. An isolated nucleic acid wherein the sequence of the isolatednucleic acid comprises a sequence at least 90% identical to a sequenceselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14, or at least 90% identical to a fragment thereof, wherein thenucleic acid comprises the sequence of a constitutive Pichia pastorispromoter.

30. The isolated nucleic acid of clause 29 wherein the sequence of theisolated nucleic acid is at least 95% identical to a sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 orat least 95% identical to a fragment thereof.

31. The isolated nucleic acid of clause 29 wherein the sequence of theisolated nucleic acid is at least 98% identical to a sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 orat least 98% identical to a fragment thereof.

32. The isolated nucleic acid sequence of clause 29 wherein the sequenceof the isolated nucleic acid is a sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or a fragmentthereof. 33. The isolated nucleic acid of any one of clauses 29 to 32operably linked to a heterologous coding sequence.

34. The isolated nucleic acid of clause 33 wherein the heterologouscoding sequence encodes a protein selected from the group consisting ofa toxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine,a structural protein, an immunogenic protein, and a cell signalingprotein.

35. The isolated nucleic acid of clause 34 wherein the protein is anenzyme for use in animal feed.

36. The isolated nucleic acid of clause 35 wherein the protein isselected from the group consisting of a phytase, a mannanase, agalactosidase, an amylase, a glucanase, a cellulase, a protease, and axylanase.

37. The isolated nucleic acid of clause 36 wherein the protein is aphytase.

38. The isolated nucleic acid of clause 36 wherein the protein is agalactosidase.

39. An expression vector comprising the isolated nucleic acid of any oneof clauses 29 to 38.

40. A host cell comprising the expression vector of clause 39.

41. A host cell comprising the isolated nucleic acid of any one ofclauses 29 to 38.

42. The host cell of any one of clauses 40 or 41 wherein the host cellis a Pichia species.

43. The host cell of clause 42 wherein the Pichia species is Pichiapastoris.

44. A DNA construct comprising the isolated nucleic acid of any one ofclauses 29 to 38.

45. A method of producing a protein, the method comprising the step of

culturing in a culture medium a host cell comprising a first expressioncassette comprising the isolated nucleic acid of any one of clauses 29to 38 operably linked to a heterologous coding sequence encoding aprotein, wherein the culturing is done under conditions permittingexpression of the protein.

46. The method of clause 45 wherein the protein is selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

47. The method of clause 46 wherein the protein is an enzyme for use inanimal feed.

48. The method of clause 47 wherein the protein is selected from thegroup consisting of a phytase, a mannanase, a galactosidase, an amylase,a glucanase, a cellulase, a protease, and a xylanase.

49. The method of clause 48 wherein the protein is a phytase.

50. The method of clause 48 wherein the protein is a galactosidase.

51. The method of any one of clauses 45 to 50 wherein the protein isexpressed using the first expression cassette in combination with asecond expression cassette.

52. The method of clause 51 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising thesequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ IDNO:16 have promoter activity, or any other AOX 1 or AOX 2 promotersequence.

53. The method of clause 51 wherein the protein is expressed using thefirst expression cassette, the second expression cassette, and a thirdexpression cassette.

54. The method of clause 53 wherein the third expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising asequence at least 90% identical to a sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, or at least 90% identical to afragment thereof.

55. The method of clause 51 wherein the second expression cassettecomprises the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising asequence at least 90% identical to a sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, or at least 90% identical to afragment thereof.

56. An isolated protein produced according to the method of any one ofclauses 45 to 55.

57. The host cell of any one of clauses 40 or 41 wherein the host cellis selected from the group consisting of Hansenula species, Pichiaspecies, Saccharomyces species, Schizosaccharomyces species, Torulasporaspecies, a Candida species, a Yarrowia species, and Kluveromycesspecies.

58. A host cell comprising the DNA construct of clause 44 wherein thehost cell is selected from the group consisting of Hansenula species,Pichia species, Saccharomyces species, Schizosaccharomyces species,Torulaspora species, a Candida species, a Yarrowia species, andKluveromyces species.

59. The method of any one of clauses 45 to 55 wherein the host cell isselected from the group consisting of Hansenula species, Pichia species,Saccharomyces species, Schizosaccharomyces species, Torulaspora species,Candida species, a Yarrowia species, and Kluveromyces species.

60. The host cell of any one of clauses 12 or 13 wherein the host cellis a methylotrophic yeast.

61. The host cell of clause 60 wherein the host cell is selected fromthe group consisting of Hansenula species, Pichia species, and Candidaspecies.

62. A host cell comprising the DNA construct of clause 16 wherein thehost cell is a methylotrophic yeast.

63. The host cell of clause 62 selected from the group consisting ofHansenula species, Pichia species, and Candida species.

64. The method of any one of clauses 17 to 27 wherein the host cell is amethylotrophic yeast.

65. The method of clause 64 wherein the host cell is selected from thegroup consisting of Hansenula species, Pichia species, and Candidaspecies.

66. The method of any one of clauses 25 or 53 wherein the thirdexpression cassette comprises the heterologous coding sequence encodingthe protein operably linked to an isolated nucleic acid having asequence comprising the sequence of SEQ ID NO:15 or SEQ ID NO:16 whereinSEQ ID NO:15 and SEQ ID NO:16 have promoter activity, or any other AOX 1or AOX 2 promoter sequence.

67. A method of producing one or more proteins, the method comprisingthe step of culturing in a culture medium a host cell comprising a firstexpression cassette, a second expression cassette, and one or moreadditional expression cassettes, wherein each of the one or moreadditional expression cassettes comprises the isolated nucleic acid ofany one of clauses 1 to 4 operably linked to a heterologous codingsequence encoding the one or more proteins, wherein the culturing isdone under conditions permitting expression of the one or more proteins.

68. A method of producing one or more proteins, the method comprisingthe step of culturing in a culture medium a host cell comprising a firstexpression cassette, a second expression cassette, and one or moreadditional expression cassettes, wherein each of the one or moreadditional expression cassettes comprises the isolated nucleic acid ofany one of clauses 29 to 38 operably linked to a heterologous codingsequence encoding the one or more proteins, wherein the culturing isdone under conditions permitting expression of the one or more proteins.

69. The method of any one of clauses 17 or 45 further comprising thestep of purifying the protein from the medium of the cultured host cell.

70. The method of any one of clauses 67 or 68 further comprising thestep of purifying one or more of the one or more proteins from themedium of the cultured host cell.

71. The isolated nucleic acid, host cell, expression vector, isolatedprotein, DNA construct, or method of any one of clauses 1 to 70 whereinthe isolated nucleic acid consists of any one of SEQ ID NOS. 1 to 14, ora fragment thereof.

72. An isolated nucleic acid consisting of any one of SEQ ID NOS. 1 to14, or a fragment thereof.

The phrase “consists of” or “consisting of” means that the sequencespecified by the SEQ ID NO. has no additional nucleotide sequences otherthan those corresponding to the SEQ ID NO.

73. The isolated nucleic acid of clause 5 wherein the heterologouscoding sequence encodes a protein or a polypeptide selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

74. The isolated nucleic acid of clause 29 wherein the heterologouscoding sequence encodes a protein or a polypeptide selected from thegroup consisting of a toxin, an antibody, a hormone, an enzyme, a growthfactor, a cytokine, a structural protein, an immunogenic protein, and acell signaling protein.

75. A method of producing a protein or a polypeptide, the methodcomprising the step of

culturing in a culture medium a host cell comprising a first expressioncassette comprising the isolated nucleic acid of any one of clauses 1 to4 operably linked to a heterologous coding sequence encoding the proteinor the polypeptide, wherein the culturing is done under conditionspermitting expression of the protein or the polypeptide.

76. A method of producing a protein or a polypeptide, the methodcomprising the step of culturing in a culture medium a host cellcomprising a first expression cassette comprising the isolated nucleicacid of any one of clauses 29 to 38 operably linked to a heterologouscoding sequence encoding the protein or the polypeptide, wherein theculturing is done under conditions permitting expression of the proteinor the polypeptide.

Any yeast expression system known to those skilled in the art can beused in accordance with the present invention. For example, variousyeast expression systems are described in U.S. Pat. Nos. 6,451,572,6,841,370, 6,974,690, 7,320,876, 7,078,035, 7,138,260, and PCTPublication No. WO 2007/112739, all incorporated herein by reference. Inany of the embodiments described herein, any of these yeast expressionsystems can be used. Alternatively, any yeast species or yeastexpression system suitable for expression of a protein can be usedincluding yeast species, such as Saccharomyces species (e.g.,Saccharomyces cerevisiae), Kluyveromyces species (e.g., Kluyveromyceslactis), Torulaspora species, Yarrowia species (e.g., Yarrowialipolitica), Schizosaccharomyces species (e.g., Schizosaccharomycespombe). In another embodiment, methylotrophic yeast species such asPichia species (e.g., Pichia pastoris or Pichia methanolica), Hansenulaspecies (e.g., Hansenula polymorpha), Torulopsis species, Komagataellaspecies, Candida species (e.g., Candida boidinii), and Karwinskiaspecies can be used, in particular when the promoter is amethanol-inducible promoter. In one embodiment the protein can beexpressed in the methylotrophic yeast Pichia pastoris. Methylotrophicyeast are capable of utilizing methanol as a sole carbon source for theproduction of the energy resources necessary to maintain cellularfunction. Methylotrophic yeast contain genes encoding enzymes formethanol utilization such as the genes encoding alcohol oxidase. Any ofthese host cells can be a host cell strain that is heterologous to thepromoter described herein (i.e., the host cell does not normally containin nature the promoter described herein).

A yeast expression system can be used to produce a sufficient amount ofthe protein intracellularly, or secreted from the yeast cells so thatthe protein can be conveniently isolated and purified from the culturemedium. As used herein, the term “expression” means transcription and/ortranslation of a nucleic acid in a host cell. A yeast expression systemmay include, but is not limited to, the yeast host cell and theexpression vector (e.g., a DNA construct) used to express the protein.The expression vector can contain a promoter described herein and, as isknown in the art, the promoter is heterologous to the expression vector(i.e., the combination does not occur in nature). In one embodiment,secretion of the protein into the culture medium is controlled by asignal peptide (e.g., the yeast α-factor signal peptide, the yeast KILM1signal peptide, the yeast PHO1 signal peptide, or the yeast SUC2 signalpeptide) incorporated into the expression vector and which is capable ofdirecting the secretion of the expressed protein out of the yeast cell.In other embodiments, other signal peptides suitable for facilitatingsecretion of the protein from yeast cells are known to those skilled inthe art. In one aspect, the signal peptide is typically cleaved from theprotein after secretion.

In various embodiments, any expression vector known to the skilledartisan (e.g., a vector that replicates autonomously or integrates intothe host genome) and compatible with a yeast expression system can beused. As used herein, the term “vector” means any plasmid, or othervector, in double-stranded or single-stranded form or in linear orcircular form that can transform a yeast cell by integration into theyeast cell genome or by existing extrachromosomally (e.g., anautonomously replicating plasmid). As is known in the art, a vector(e.g., expression vector or expression cassette) is a nucleic acidconstruct used to transform a host cell for expression of a protein,polypeptide, or peptide and the vector is not found in nature in thehost cell it transforms.

In one embodiment, the expression vector has restriction endonucleasecleavage sites for the insertion of DNA fragments (e.g., one or morecloning sites and/or a multiple cloning site), and genetic markers forselection of transformants. For example, the genetic markers forselection of transformants can include a selection marker that allows atransformed yeast to grow on a medium devoid of a necessary nutrientthat cannot be produced by a deficient strain, a selection marker thatencodes an enzyme for which chromogenic substrates are known, or aselection marker that provides resistance to a drug, including, but notlimited to, G418, Nourseothricin (Nat), Zeocin, Blasticidin, orHygromycin. In another embodiment, the expression vector has aterminator sequence for transcription termination (e.g., the AOX 1 orHSP150 terminator). In another embodiment, the expression vector has a3′ untranslated region downstream from the protein coding sequence witha polyadenylation site. As used herein, “3′ untranslated region” meansnucleotide sequences that are not translated into protein and arelocated downstream from a coding sequence for a protein. Typically, a 3′untranslated region includes regulatory sequences for mRNA processing.In another embodiment, the expression vector has an origin ofreplication (e.g., a bacterial origin of replication) for use insynthesizing and amplifying the vector, for example, in a bacterialhost. Various expression vectors are described in U.S. Pat. Nos.6,451,572, 6,841,370, 6,974,690, 7,320,876, 7,078,035, 7,138,260, andPCT Publication No. WO 2007/112739, all incorporated herein byreference. The construction and use of expression vectors is describedin Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rdEdition, Cold Spring Harbor Laboratory Press, (2001), incorporatedherein by reference. In another embodiment, the expression vector, or afragment thereof, can be synthesized de novo or PCR can be used toamplify and join sections of expression vectors.

As used herein, “regulatory sequences” means nucleotide sequences thatare typically upstream or downstream from the 5′ or 3′ end,respectively, of a protein coding sequence. Regulatory sequences are nottranslated into protein. Regulatory sequences include, but are notlimited to, sequences that affect RNA processing or stability, such aspolyadenylation signal sequences, enhancers, repressor binding sites,and promoters.

In one embodiment, the protein coding sequence can be operably linked inthe expression vector to the promoter sequence capable of directing theexpression of the protein, for example, in yeast. As used herein,“operably linked” means functionally linked. As described herein, thepromoter can be a constitutive or an inducible promoter, such as amethanol inducible promoter. As used herein, a “constitutive promoter”means a promoter that regulates expression of a gene of interest. Theterm “constitutive promoter” is known in the art. As used herein, an“inducible promoter” means a regulated promoter that is turned on in acell by an external stimulus, such as a chemical, light, a change intemperature, a change in cell density, or a protein, such as a hormone.A methanol inducible promoter can have some constitutive activity,although a methanol inducible promoter has maximal activity when inducedin the presence of methanol. Likewise, a constitutive promoter can havesome inducible activity, but a “constitutive promoter” as used hereindoes not have maximal activity when induced in the presence of methanol.

As used herein “promoter” means a nucleotide sequence typically locatedupstream from the 5′ end of a coding sequence for a protein thatcontrols the transcription of RNA from DNA, in part, by interacting withvarious regulatory factors that control transcription. In oneembodiment, the promoter may be derived from the same species of yeastas the yeast host cell used for protein expression. In anotherembodiment, the promoter may be derived from a different yeast speciesthan the yeast host cell used for protein expression. In one embodiment,a promoter may include a TATA box sequence that acts as a recognitionsite to direct initiation of transcription, including, but not limitedto one or more transcriptional enhancer elements. The enhancer elementsmay be proximal or distal to the TATA box sequence and may be in anormal 5′ to 3′ orientation or may be in a 3′ to 5′ orientation. Inanother embodiment, an enhancer element may be an enhancer elementnative to the promoter sequence or it may be a heterologous enhancerelement inserted into the expression vector construct. An “enhancerelement” as used herein is a regulatory element that can stimulatepromoter activity.

In various illustrative embodiments described herein, the promoter canbe an isolated nucleic acid wherein the sequence of the isolated nucleicacid comprises a sequence at least 80%, at least 85%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6, or at least 80%, at least 85%, at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a fragment thereof, wherein the isolated nucleic acidcomprises the sequence of a methanol-inducible Pichia pastoris promoter.In another embodiment, the isolated nucleic acid sequence is a sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a fragment thereofwherein the isolated nucleic acid comprises the sequence of amethanol-inducible Pichia pastoris promoter.

In other embodiments, the promoter can be an isolated nucleic acidwherein the sequence of the isolated nucleic acid comprises a sequenceat least 80%, at least 85%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, andSEQ ID NO:14 or at least 80%, at least 85%, at least 90%, at least 92%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a fragment thereof, wherein the isolated nucleic acidcomprises the sequence of a constitutive Pichia pastoris promoter. Inanother embodiment, the isolated nucleic acid sequence is a sequenceselected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14, or a fragment thereof wherein the isolated nucleic acid comprisesthe sequence of a constitutive Pichia pastoris promoter.

As used herein “an isolated nucleic acid” means a nucleic acid that issubstantially free of sequences that naturally flank the nucleic acid inthe genomic DNA of the organism from which the nucleic acid is derived.For example, in various embodiments, an isolated nucleic acid inaccordance with the invention can contain less than about 2 kb, lessthan about 1 kb, less than about 0.5 kb, less than about 0.1 kb ofnucleotide sequences, less than about 0.05 kb, or no nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the organism from which the isolated nucleic acid is derived.

As used herein, “a fragment thereof” when referring to the isolatednucleic acid molecule means a fragment of the isolated nucleic acid ofSEQ ID NOS: 1 to 14. In various illustrative embodiments, the fragmentcan be about 50 nucleotides in length, about 100 nucleotides in length,about 200 nucleotides in length, about 300 nucleotides in length, about400 nucleotides in length, about 500 nucleotides in length, about 600nucleotides in length, about 700 nucleotides in length, about 800nucleotides in length, or about 900 nucleotides in length. In otherembodiments, the fragment can extend about 50 nucleotides, about 100nucleotides, about 200 nucleotides, about 300 nucleotides, about 400nucleotides, about 500 nucleotides, about 600 nucleotides, about 700nucleotides, about 800 nucleotides, or about 900 nucleotides upstreamfrom the 3′ end of the isolated nucleic acid of any of SEQ ID NOS: 1 to14. In yet other embodiments, the fragment can include about 50nucleotides, about 100 nucleotides, about 200 nucleotides, about 300nucleotides, or about 350 nucleotides upstream and/or downstream fromthe TATA box sequence found in each of SEQ ID NOS: 1 to 14.

In various embodiments, the isolated nucleic acids described herein maybe purified by techniques for purification of nucleic acids (e.g., DNA)that are well-known in the art. For example, the nucleic acids may beseparated from contaminants by physical methods including, but notlimited to, centrifugation, pressure techniques, or by using a substancewith affinity for nucleic acids (e.g., DNA), such as, for example,silica beads. After sufficient washing, the isolated nucleic acids maybe suspended in either water or a buffer. In other embodiments,commercial kits are available, such as Qiagen™, Nuclisensm™, and Wizard™(Promega), and Promegam™. Methods for purifying nucleic acids aredescribed in Sambrook et al., “Molecular Cloning: A Laboratory Manual”,3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporatedherein by reference. An isolated nucleic acid as described herein may bean isolated nucleic acid that is also purified, or the isolated nucleicacid may be impure. A “purified nucleic acid” is substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

The isolated nucleic acids described herein are capable of specifichybridization, under appropriate hybridization conditions (e.g.,appropriate buffer, ionic strength, temperature, formamide, and MgCl₂concentrations), to a complementary nucleic acid. The isolated nucleicacids described herein can be modified by substitution, deletion,truncation, and/or can be fused with other nucleic acid moleculeswherein the resulting isolated nucleic acids hybridize specifically tothe complemetary nucleic acids.

Also within the scope of the invention are nucleic acids complementaryto the isolated nucleic acids, or fragments thereof, described herein,and those that hybridize to the isolated nucleic acids described hereinor those that hybridize to their complements under highly stringentconditions. As used herein, the term “complementary” refers to theability of purine and pyrimidine nucleotide sequences to associatethrough hydrogen bonding to form double-stranded nucleic acid molecules.Guanine and cytosine, adenine and thymine, and adenine and uracil arecomplementary and can associate through hydrogen bonding resulting inthe formation of double-stranded nucleic acid molecules when two nucleicacid molecules have “complementary” sequences. The complementary DNAsequences are referred to as a “complement.”

In accordance with the invention “highly stringent conditions” meanshybridization at 65° C. in 5×SSPE and 50% formamide, and washing at 65°C. in 0.5×SSPE. Conditions for high stringency hybridization aredescribed in Sambrook et al., “Molecular Cloning: A Laboratory Manual”,3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporatedherein by reference. In some illustrative aspects, hybridization canoccur along the full-length of the isolated nucleic acid, or along partof its length, or to a fragment thereof.

Also included are isolated nucleic acid molecules having about 60%,about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about95%, 96%, 97%, 98%, and 99% identity to the isolated nucleic acidsdescribed herein, or to a fragment thereof. Determination of percentidentity or similarity between sequences can be done, for example, byusing the GAP program (Genetics Computer Group, software; now availablevia Accelrys on http://www.accelrys.com), and alignments can be doneusing, for example, the ClustalW algorithm (VNTI software, InforMaxInc.). A sequence database can be searched using the isolated nucleicacid sequence of interest, or the sequence of a fragment thereof.Algorithms for database searching are typically based on the BLASTsoftware (Altschul et al., 1990). In some embodiments, the percentidentity can be determined along the full-length of the isolated nucleicacid.

Techniques for synthesizing the isolated nucleic acids described hereinare well-known in the art and include chemical syntheses and recombinantmethods. Such techniques are described in Sambrook et al., “MolecularCloning: A Laboratory Manual”, 3rd Edition, Cold Spring HarborLaboratory Press, (2001), incorporated herein by reference. Isolatednucleic acid molecules can also be made commercially. Techniques forsynthesizing the isolated nucleic acids described herein are well-knownin the art. The isolated nucleic acids described herein can be analyzedby techniques known in the art, such as restriction enzyme analysis orsequencing, to determine the sequence of the isolated nucleic acids.Thus, isolated nucleic acids described herein may be synthetic.

In illustrative aspects, yeast cells are transformed with an expressionvector comprising a heterologous nucleic acid encoding the protein ofinterest operably linked to one of the promoters described herein usingprocedures well-known to those skilled in the art. The term“transformation” means the transfer of a nucleic acid, or a nucleic acidfragment, into a host cell. In illustrative embodiments, suchtransformation protocols include electroporation, lithium acetatemethods, and use of spheroplasts. In illustrative aspects, the expressednucleic acid coding sequence can be a heterologous nucleic acid codingsequence. As used herein, a heterologous coding sequence is defined asan artificial or synthetic nucleic acid or a nucleic acid originatingfrom a different species than the species from which the promotersequence was derived. Thus, a heterologous coding sequence linked to apromoter described herein does not occur in nature.

In various embodiments, the transformed yeast cells may be grown bytechniques including batch and continuous fermentation in a liquidmedium or on a semi-solid medium, or a solid medium. Typically,“conditions permitting expression of the protein” as used herein meansconditions for batch or continuous fermentation of yeast in a liquidmedium, but growth on a semi-solid medium, such as agar, is notexcluded. Culture media for yeast cells are known in the art and aretypically supplemented with a carbon source (e.g., glucose). A typicalyeast culture medium is YPD broth (Sunrise Science Products, Inc.)comprising yeast extract (10 grams), Bacto peptone (20 grams), anddextrose (20 grams). In one illustrative aspect, the transformed yeastcells can be grown aerobically at 30° C. in a controlled pH environment(a pH of about 6) and with the carbon source (e.g., glucose) maintainedcontinuously at a predetermined level known to support growth of theyeast cells to a desired density within a specific period of time.

In one illustrative embodiment, a method of producing a protein isprovided. The method comprises the step of culturing in a culture mediuma host cell comprising a first expression cassette comprising anisolated nucleic acid of any one of SEQ ID NOS: 1 to 14, or a fragmentthereof, operably linked to a heterologous coding sequence encoding aprotein, wherein the culturing is done under conditions permittingexpression of the protein. The method can further comprise the step ofpurifying the protein from the medium of the cultured host cell. As usedherein, an “expression cassette” means the elements of an expressionvector that direct the yeast cell to make RNA. An expression cassettecomprises at least regulatory sequences (e.g., a promoter) and a codingsequence for the RNA and protein (i.e., an open reading frame). Theisolated nucleic acid can be at least 80%, at least 85%, at least 90%,92%, 95%, 96%, 97%, 98%, or 99% homologous to the isolated nucleic acidof any of SEQ ID NOS: 1 to 14, or a fragment thereof. In variousillustrative aspects, the protein coding sequence can be from abacterium, a yeast, a fungus, or a virus.

In this method embodiment, the protein can be expressed using the firstexpression cassette in combination with a second expression cassette. Inanother embodiment, the second expression cassette can comprise 1) theheterologous coding sequence encoding the protein operably linked to anisolated nucleic acid having a sequence comprising the sequence of SEQID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ ID NO:16 havepromoter activity, or any other known methanol-regulated promoter, suchas AOX 1, AOX 2, FLD, or DAS promoter sequences, or 2) the isolatednucleic acid of any one of SEQ ID NOS: 1 to 14, or a fragment thereof,operably linked to the heterologous coding sequence encoding theprotein.

In yet another embodiment, the protein can be expressed using the firstexpression cassette, the second expression cassette, and a thirdexpression cassette. In another illustrative aspect, the thirdexpression cassette can comprise 1) the heterologous coding sequenceencoding the protein operably linked to an isolated nucleic acid havinga sequence comprising the sequence of SEQ ID NO:15 or SEQ ID NO:16wherein SEQ ID NO:15 and SEQ ID NO:16 have promoter activity, or anyother known methanol-regulated promoters, such as AOX 1, AOX 2, FLD, orDAS promoter sequences, or 2) the isolated nucleic acid of any one ofSEQ ID NOS: 1 to 14, or a fragment thereof, operably linked to theheterologous coding sequence encoding the protein.

In another embodiment, any number of additional expression cassettes canbe used and the expression cassettes can comprise 1) the heterologouscoding sequence encoding the protein operably linked to an isolatednucleic acid having a sequence comprising the sequence of SEQ ID NO:15or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ ID NO: have promoteractivity, or any other known methanol-regulated promoters, such as AOX1, AOX 2, FLD, or DAS promoter sequences, or 2) the isolated nucleicacid of any one of SEQ ID NOS: 1 to 14, or a fragment thereof, operablylinked to the heterologous coding sequence encoding the protein. Inanother embodiment, the first expression cassette, the second expressioncassette, and the third expression cassette as described above are usedin the method, along with a fourth expression cassette, a fifthexpression cassette, and a sixth expression cassette wherein all of theexpression cassettes comprise the isolated nucleic acid of any one ofSEQ ID NOS: 1 to 14, or a fragment thereof, operably linked to theheterologous coding sequence encoding the protein.

In still another embodiment, a method of producing one or more proteinsis provided. The method comprises the step of culturing in a culturemedium a host cell comprising a first expression cassette, a secondexpression cassette, and, optionally, one or more additional expressioncassettes, wherein each of the expression cassettes comprises 1) aheterologous coding sequence encoding the one or more proteins operablylinked to an isolated nucleic acid having a sequence comprising thesequence of SEQ ID NO:15 or SEQ ID NO:16 wherein SEQ ID NO:15 and SEQ IDNO:16 have promoter activity, or any other known methanol-regulatedpromoters, such as AOX 1, AOX 2, FLD, or DAS promoter sequences, or 2)the isolated nucleic acid of any one of SEQ ID NOS: 1 to 14, or afragment thereof, operably linked to a heterologous coding sequenceencoding the one or more proteins, wherein the culturing is done underconditions permitting expression of the one or more proteins. The methodcan further comprise the step of purifying one of the one or moreproteins from the medium of the cultured host cell.

In any of the embodiments described in the preceding two paragraphs, theisolated nucleic acid can be at least 80%, at least 85%, at least 90%,92%, 95%, 96%, 97%, 98%, or 99% homologous to the isolated nucleic acidof any of SEQ ID NOS: 1 to 14, or a fragment thereof. In any of theembodiments described in the preceding two paragraphs, the expressioncassettes can be included in one expression vector or in multipleexpression vectors. In any of the embodiments described in the precedingtwo paragraphs, the expression vectors into which the expressioncassettes are incorporated can be vectors that replicate autonomously orthat integrate into the host cell genome.

In various illustrative embodiments, the protein encoded by the any ofthe heterologous coding sequences described herein operably linked tothe promoter can be a protein selected from the group consisting of atoxin, an antibody, a hormone, an enzyme, a growth factor, a cytokine, astructural protein, an immunogenic protein (e.g., a vaccine antigen),and a cell signaling protein. In one embodiment, the protein is anenzyme for use in animal feed. In this embodiment, the protein can beselected from the group consisting of a phytase, a mannanase, agalactosidase, an amylase, a glucanase, a cellulase, a protease, and axylanase. In another embodiment, the protein can be an enzyme useful forglycosylation. In another aspect, more than one protein can be expressedby using multiple expression cassettes, each with a heterologous codingsequence, operably linked to a promoter. However, these protein examplesare non-limiting and any protein, polypeptide, or peptide capable ofbeing expressed in yeast can be expressed in accordance with theisolated nucleic acids, expression vectors, host cells, DNA constructs,methods, and isolated proteins described herein. The enzymes describedabove can be from any species (e.g., fungal species, such as a yeastspecies).

The yeast-expressed proteins for use in accordance with the presentinvention can be produced in purified form by conventional techniques.As used herein, “isolated protein” means a purified protein. A purifiedprotein is substantially free from other yeast cell contaminants orcontaminants from the culture medium. For example, “substantially free”from other yeast cell contaminants or contaminants from the culturemedium means that the protein is at least about 60% pure, at least about70% pure, at least about 80% pure, at least about 90% pure, at leastabout 95% pure, at least about 98% pure, about 60% pure, about 70% pure,about 80% pure, about 90% pure, about 95% pure, or about 98% pure (allbased on dry weight). Typically, the protein is secreted into the yeastculture medium and is collected from the culture medium.

In one illustrative embodiment, for purification from the culture mediumthe protein can, for example, be subjected to ammonium sulfateprecipitation followed by DEAE-Sepharose column chromatography. In otherembodiments, conventional techniques known to those skilled in the artcan be used such as ammonium sulfate or ethanol precipitation, acidextraction, gel filtration, anion or cation exchange chromatography,DEAE-Sepharose column chromatography, hydroxylapatite chromatography,lectin chromatography, affinity chromatography, solvent-solventextraction, ultrafiltration, and HPLC.

Alternatively, purification steps may not be required because theprotein may be present in such high concentrations in the culture mediumthat the protein is essentially pure in the culture medium (e.g., 70 to80% pure). In one embodiment, the protein is collected from the culturemedium without further purification steps by chilling the yeast culture(e.g., to about 4° C. to about 8° C.) and removing the yeast cells usingsuch techniques as centrifugation, microfiltration, and rotary vacuumfiltration. The protein in the cell-free medium can then be concentratedby such techniques as, for example, ultrafiltration and tangential flowfiltration.

In some embodiments where the protein is not secreted into the culturemedium, the yeast cells can be lysed, for example, by sonication, heat,or chemical treatment, and the homogenate centrifuged to remove celldebris. The supernatant can then be subjected to ammonium sulfateprecipitation, and additional fractionation techniques as required, suchas gel filtration, ion exchange chromatography, DEAE-Sepharose columnchromatography, affinity chromatography, solvent-solvent extraction,ultrafiltration, and HPLC to purify the protein. It should be understoodthat the purification methods described above for purification ofproteins from the culture medium or from lysed yeast cells arenon-limiting and any purification techniques known to those skilled inthe art can be used to purify the yeast-expressed protein if suchtechniques are required to obtain a substantially pure protein.

Various formulations of the purified protein preparations may beprepared in accordance with the invention. In some embodiments, theproteins can be stabilized through the addition of other proteins (e.g.,gelatin and skim milk powder), chemical agents (e.g., glycerol,polyethylene glycol, EDTA, potassium sorbate, sodium benzoate, andreducing agents and aldehydes), polysaccharides, monosaccharides, lipids(hydrogenated vegetable oils), and the like. In one embodiment, proteinsfor addition to food products or animal feed blends can be dried (e.g.,spray drying, drum drying, and lyophilization) and formulated aspowders, granules, pills, mineral blocks, liquids, and gels throughknown processes. In one embodiment, gelling agents such as gelatin,alginate, collagen, agar, pectin and carrageenan can be used.

In alternate embodiments, the protein expression can be forintracellular expression, such as for enzymatic action in the yeast in abiotransformation process, or for display on the yeast cell surface. Forsuch embodiments, the protein, expressed as described herein, is notpurified.

In various embodiments, the proteins described above are selected fromthe group consisting of a toxin, an antibody, a hormone, an enzyme, agrowth factor, a cytokine, a structural protein, an immunogenic protein(e.g., a vaccine antigen), and a cell signaling protein. In anotherembodiment, the proteins described above are selected from the groupconsisting of an antibody, a hormone, an enzyme, a growth factor, acytokine, a structural protein, an immunogenic protein (e.g., a vaccineantigen), and a cell signaling protein. In yet another embodiment thecoding sequence for a protein, a fragment thereof, a fusion protein(e.g., a chimeric protein), or a peptide, can be used in accordance withthe invention. In another embodiment, a modified protein can beexpressed, such as a mutated protein or a protein with non-natural aminoacids.

In one embodiment, the toxins can be proteins such as, for example,botulinum toxin or verotoxin-1, and after preparation using the methods,isolated nucleic acids, expression vectors, host cells, and DNAconstructs described herein, the toxins can be modified using atargeting agent so that they are directed specifically to diseasedcells. In another illustrative aspect, the antibody can be a humanizedantibody, an antibody that is not humanized, a nanobody, or an antibodyfragment, such as an Fab fragment of an antibody or a single-chainantibody. In another embodiment, the hormone can be, for example, agonadotropin, an adrenocorticotrophic hormone, a growth hormone,vasopressin, oxytocin, somatostatin, gastrin, or leptin. In anotherillustrative embodiment, the growth factor can be insulin, epidermalgrowth factor, fibroblast growth factor, vascular endothelial growthfactor, erythropoietin, platelet-derived growth factor, thrombopoietin,or a bone morphogenic protein. In one aspect, the cytokine can be IL-2,IFN-α, IFN-γ, or GM-CSF. In another illustrative aspect, the vaccineproteins can be any suitable vaccine proteins that are immunogenic in apatient or an animal, including, but not limited to, HPV proteins (e.g.,HPV 16 and HPV 18), and tetanus vaccine proteins, as examples. Inanother illustrative embodiment, the enzymes can be, for example,enzymes for animal feeds as discussed herein, acetylcholinesterase, orcyclooxygenase, or any other useful enzyme that can be expressed inyeast. In another embodiment, structural proteins can be expressed, forexample, netrins, actin-binding proteins, or myosin, and, in anotherembodiment, cell signaling proteins such as ras proteins, kinases, theErbB2 protein (the Her-2 receptor) can be expressed using the methods,isolated nucleic acids, expression vectors, host cells, and DNAconstructs described herein.

In one embodiment, the protein is an enzyme for use in animal feed. Inthis embodiment, the protein can be selected from the group consistingof a phytase, a mannanase, a galactosidase, an amylase, a glucanase, acellulase, a protease, and a xylanase, or a combination thereof. Forexample, a variety of phytases may be expressed according to the methodsdescribed herein. Exemplary of phytase genes (i.e., a phytase codingsequence) that can be expressed in accordance with the invention arephytase genes derived from bacteria, filamentous fungi, plants, andyeast, such as the appA (Gene Bank accession number M58708) and appA2(Gene Bank accession number 250016) genes derived from Escherichia coliand the phyA and phyB genes derived from the fungus Aspergillus niger,or any mutant of these genes that retains or has improved myo-inositolhexakisphosphate phosphohydrolase activity (see, for example, Rodriguezet al., Arch. of Biochem. and Biophys. 382: 105-112 (2000), incorporatedherein by reference). Substituted, deleted, and truncated phytase genes,or a fragment thereof, can also be expressed in accordance with theinvention.

In one embodiment, the protein expressed using the methods describedherein can be used in animal feed comprising an animal feed blend. Invarious embodiments, any animal feed blend known in the art can be usedsuch as rapeseed meal, cottonseed meal, soybean meal, and cornmeal.Optional ingredients of the animal feed blend include sugars and complexcarbohydrates such as both water-soluble and water-insolublemonosaccharides, disaccharides and polysaccharides. Optional amino acidingredients that can be added to the feed blend are arginine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan, valine, tyrosine ethyl HCl, alanine, aspartic acid, sodiumglutamate, glycine, proline, serine, cysteine ethyl HCl, and analogs,and salts thereof. Vitamins that can be optionally added are thiamineHCl, riboflavin, pyridoxine HCl, niacin, niacinamide, inositol, cholinechloride, calcium pantothenate, biotin, folic acid, ascorbic acid, andvitamins A, B, K, D, E, and the like. Minerals, protein ingredients,including protein obtained from meat meal or fish meal, liquid orpowdered egg, fish solubles, whey protein concentrate, oils (e.g.,soybean oil), cornstarch, calcium, inorganic phosphate, copper sulfate,salt, and limestone can also be added. Antioxidants can also be added.

In another embodiment, a kit comprising an expression vector comprisingthe isolated nucleic acid of SEQ ID NO: 1 to SEQ ID NO: 14, or afragment thereof, is provided. In one illustrative aspect, the isolatednucleic acid, or the fragment, can be 80%, 85%, 90%, 92%, 95%, 96%, 97%,98%, or 99% identical to the isolated nucleic acid of SEQ ID NO: 1 toSEQ ID NO: 14, or to the fragment thereof. In various illustrativeembodiments, the fragment can be about 50 nucleotides in length, about100 nucleotides in length, about 200 nucleotides in length, about 300nucleotides in length, about 400 nucleotides in length, about 500nucleotides in length, about 600 nucleotides in length, about 700nucleotides in length, about 800 nucleotides in length, or about 900nucleotides in length. In other embodiments, the fragment can extendabout 50 nucleotides, about 100 nucleotides, about 200 nucleotides,about 300 nucleotides, about 400 nucleotides, about 500 nucleotides,about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, orabout 900 nucleotides upstream from the 3′ end of the isolated nucleicacid of any of SEQ ID NOS: 1 to 14. In yet other embodiments, thefragment can include about 50 nucleotides, about 100 nucleotides, about200 nucleotides, about 300 nucleotides, or about 350 nucleotidesupstream and/or downstream from the TATA box sequence found in each ofSEQ ID NOS: 1 to 14.

In one embodiment, the expression vector (i.e., with the heterologouspromoter) included in the kit can have any of the other elementsdescribed herein, such as a selection marker, a cloning site, such as amultiple cloning site, an enhancer, a termination sequence, a signalpeptide sequence, and the like. In another aspect, the expression vectorcan be a vector that replicates autonomously or integrates into the hostcell genome. In another embodiment, the expression vector can becircularized or linearized (i.e., digested with a restriction enzyme sothat a gene of interest can easily be cloned into the expressionvector). In another embodiment, the kit can include an expression vectorand a control open reading frame encoding a marker or control gene forexpression (e.g., an open reading frame encoding a LacZ-α fragment) foruse as a control to show that the expression vector is competent to beligated and to be used with a gene of interest.

In another illustrative embodiment, the kit can contain a tubecontaining a circular expression plasmid. In another embodiment, the kitcan contain a tube with a linear expression vector that is digested witha restriction enzyme so it is ready to clone a gene of interest. In oneembodiment, the tube can be sterilized. In either of these embodiments,the kit can also include a circular or linear expression vector and acontrol open reading frame encoding a marker or control gene forexpression (e.g., an open reading frame encoding a LacZ-α fragment) foruse as a control in showing that the expression vector can be ligatedwith a gene of interest, and/or to show that the host cell is competentfor transformation.

In yet another embodiment, the kit can contain multiple differentexpression vectors. In another embodiment, the multiple differentexpression vectors can contain the same promoter, but differentselectable markers, such as genes for resistance to the drugs G418,Nourseothricin (Nat), Zeocin, Blasticidin, or Hygromycin. In thisembodiment, the kit may also contain aliquots of the drugs (e.g., G418,Nourseothricin (Nat), Zeocin, Blasticidin, or Hygromycin) in tubes, orother containers, separate from the corresponding vectors.

In any of the kit embodiments described above, the isolated nucleic acidcan consist of any one of SEQ ID NOS. 1 to 14, or a fragment thereof.The phrase “consists of” means that the sequence specified by the SEQ IDNO. has no additional nucleotide sequences other than thosecorresponding to the SEQ ID NO.

In another illustrative aspect, the kit can include other components foruse with the expression vector, such as components for transformation ofyeast cells, restriction enzymes for incorporating a protein codingsequence of interest into the expression vector, ligases, components forpurification of expression vector constructs, buffers (e.g., a ligationbuffer), instructions for use (e.g., to facilitate cloning), and anyother components suitable for use in a kit for making and using theexpression vectors described herein. In another embodiment, theexpression vector or any other component of the kit can be included inthe kit in a sealed tube (e.g., sterilized or not sterilized) or anyother suitable container or package (e.g., sterilized or notsterilized). The kits described in the preceding paragraphs that includethe expression vector comprise the expression vector comprising apromoter described herein operably linked to the vector which isheterologous to the promoter (i.e., the combination does not occur innature).

The following examples provide illustrative methods for carrying out thepractice of the present invention. As such, these examples are providedfor illustrative purposes only and are not intended to be limiting.

EXAMPLES Example 1 Promoter Expression Levels

TABLE 1 Fermentation Samples glycerol 6 hr 2 hr methanol 6 hr methanol18 hr methanol 48 hr methanol 66 hr methanol Gene starvation glycerolfeed induction induction induction induction induction AOX1 958 54 2180617607 10035 581 911 AOX2 185 28 10035 1374 1697 64 119 CAM1 316 16613201 13201 24457 36959 36959 FLD1 3405 1293 11173 10632 13473 2180614995 GPM2 884 549 7280 8614 8357 14995 17607 PP7435_Chr1-1351 2102 14912171 13473 14995 17607 11173 PP7435_Chr1-0269 7766 8636 3252 3772 66159483 5969 PP7435_Chr2-0207 13473 17607 1520 858 4496 3914 3742PP7435_Chr2-0208 21806 28506 2298 1136 7155 7280 8949 PP7435_Chr2-07902457 356 7829 7502 6433 2064 822 PP7435_Chr2-0809 5566 4305 1668 42054799 6758 2899 PP7435_Chr3-0476 6758 9425 1648 5566 6104 10632 10035PP7435_Chr3-0842 243 134 8614 6987 5684 7155 12171 PP7435_Chr4-0069 750212171 1746 1162 7829 9425 13473 PP7435_Chr4-0800 5064 5916 2382 54155415 6987 3964 SPI1 275 203 673 376 267 164 195 TDH1 8949 841 2102 9512965 813 104 TEF2 7155 2248 4305 7829 10632 3246 757 THI4 1668 68 1499512171 2542 429 419 * All numbers as transcripts per million

TABLE 2 Shake Flask Samples strain 1: strain 2: strain 3: strain 4:strain glycerol glycerol glycerol glycerol 5: shake shake shake shakemethanol Gene flask flask flask flask induction AOX1 7 8 24 22 3167 AOX212 16 9 8 109 CAM1 537 513 661 789 24110 FLD1 762 743 357 1202 17607GPM2 303 352 699 561 9425 PP7435_Chr1-1351 9 10 15 19 10035PP7435_Chr1-0269 5587 5535 11983 10362 6375 PP7435_Chr2-0207 2226 21523466 3246 680 PP7435_Chr2-0208 3167 3121 7502 4402 864 PP7435_Chr2-079041 48 31 26 1877 PP7435_Chr2-0809 6542 6433 2965 4305 6758PP7435_Chr3-0476 28506 21806 28506 28506 7829 PP7435_Chr3-0842 14 16 4844 1171 PP7435_Chr4-0069 4887 5684 8141 9425 6542 PP7435_Chr4-0800 2180617607 10035 21806 7502 SPI1 2500 2035 4799 1529 136 TDH1 1770 5415 11771095 2654 TEF2 17607 28506 3914 3803 12171 THI4 10 58 40 30 452 * Allnumbers as transcripts per million

Example 2 Strain Growth for RNA Isolation to Examine Promoter ExpressionLevels

The BG10 strain was maintained as patches on YPD Agar plates. Fortranscriptome analysis, 50 ml cultures of the strain were inoculatedfrom a patch and grown in BMGY at 30° C. (200 rpm) for approximately 16hours. The stationary culture was diluted 100-fold into fresh BMGYmedium and grown at 30° C. (200 rpm) for 6 hours. This time point wasconsidered exponential growth with glycerol as the carbon source.Aliquots were spun down in 15 ml tubes. Thereafter, supernatants werediscarded and the cell pellets were rapidly frozen in liquid nitrogen.Cell pellets were stored at −80° C. for subsequent total RNA isolation.

For RNA-Seq analysis during fermentation, a shake flask culture grown asdescribed above was expanded into a 1-liter fermentor using standardmethanol induction conditions. Cell samples were taken at various timepoints before, during, and at the end of the methanol induction. Cellpellets were collected and frozen in liquid nitrogen. Cell pellets werestored at −80° C. for subsequent total RNA isolation.

Example 3 Total RNA Isolation

FastRNA SPIN kits (MP Bio) were used to isolate total RNA. Cell lysiswas performed using a BioSpec Mini-Beadbeater 96. Total RNA was elutedfrom the spin column in 15 μl of RNase/DNase—free water, frozen inliquid nitrogen and stored at −80° C. RNA samples were shipped on dryice for RNA-Seq analysis on an Illumina HiSeq machine. RNA samples wereanalyzed using an Agilent BioAnalyzer, and all showed intact yeastribosomal RNA peaks.

Example 4 RNA Library Generation and Sequencing

mRNA libraries were prepared using Illumina reagents. A TruSeq RNASample Preparation Kit was used to selectively generate bar-coded cDNAfrom polyA RNA. After bar-coding and amplification, a total of 12samples were pooled (8 fermentation samples, 4 shake flask samples) foranalysis. Fifty base, single end reads were performed. Data was suppliedto BioGrammatics in standard FASTQ format. Reads were trimmed based onambiguous bases and quality score and then filtered to eliminate alltrimmed reads that were less than 40 bases in length. Approximately 0.3%of reads were removed from each data set.

Example 5 RNA-Seq Analysis

Data sets from an Illumina HiSeq machine were imported into CLC GenomicsWorkbench (version 6). The standard software tools from CLC GenomicsWorkbench were used for RNA-Seq analysis. RNA reads were mapped onto areference Pichia pastoris genome. Transcription profiles of 5202annotated genes were generated across the 12 sample data sets. Based onthe transcription profiles, genes with either constitutive or methanolregulated transcription patterns were identified.

Example 6 Protein Expression Analysis

Multiple promoters were inserted into a Promoter Tester Vector,(reporter plasmid, pJ-G-Agal, FIG. 17) to test protein expression.Briefly, an alpha-galactosidase (A-gal) open reading frame (ORF) in thepJ-G-A-gal is used to measure the expression level from a promoterinserted just 5′ of the A-gal ORF. Select DNA primers (IDT-DNA, table 3)were used to PCR amplify isolated DNA from Pichia genomic DNA (gDNA,BioGrammatics strain wild type Pichia pastoris strain Bg10).

TABLE 3  PCR primers for promoters. Primer position SEQ on the Promo- IDForward Promoter ter NO: primer Primer sequence sequence SAM  111231-F685 NNNGGTCTCNATCCACGAG −685 TTTCTGGACCGTATC SAM1  1 11231-F447NNNGGTCTCTATCCGGAAA −447 ACGTTAAGAGATG SAM1  1 11231-F3  NNNGGTCTCTATCCCTCTA −532 CTAAATTGCCCCAAGTG Chr1-  2 1351-F1 NNNGGTCTCNATCCGATGG −606 1351 AGACTCAGTATGATGGGGC Chr1-  2 1351-F2 NNNGGTCTCNATCCAGTAT −595 1351 GATGGGGCAAGGAAAACG THI4  3 THI4-F1 NNNGGTCTCNATCCTGGAG −404 ACCCTTAACAGGTCG GPM2  4 GPM2-F1 NNNGGTCTCNATCCGTTGG −637 GAACTGTGCCTG Chr2-  5 790-F1NNNGGTCTCNATCCACAGTG −543 0790 GTAGGTCCAACTTGG Chr3-  6 842-F1NNNCGTCTCNATCCGTAGTA −635 0842 GCCTCTCCAGCCTG Chr1-  7 269-F1NNNGGTCTCNATCCTGAAGC −611 0269 CCCTGCAACTACAGAG Chr2-  8 207-F1NNNGGTCTCNATCCGTAGAC −632 0207 GACATCCAGAGAAGTAACAG Chr2-  9 208-F1NNNGGTCTCNATCCTCAGGT −618 0208 CAGTCTTGAAGTCCTGAG Chr2- 10 809-F1NNNGGTCTCNATCCTGTGGA −653 0809 ATTCCAAAGAAGGGG Chr4- 11 069-F1NNNGGTCTCNATCCGTCCGT −467 0069 GATGTAAAATGAGACTAC Chr4- 12 800-F1NNNGGTCTCNATCCAGTCAA −490 0800 CTGGGAGCTACGGT TEF2 13 TEF2-FF1NNNGGTCTCNATCCGATGTG −729 AGGATGCGCTC Chr3- 14 476-F1NNNGGTCTCNATCCTCAATG −650 0476 ACCACGGTAACATGAAAAC GAP GAP-F1NNNGGTCTCNATCCAATGGA −619 CCAAATTGTTGCAAGGT DAS 07226F620NNNGGTCTCCATCCCTTTGT −382 TGAGCAACA DAS 07226F518 NNNGGTCTCGATCCGCCCAA−483 ACGAACAG AOX AOXF1 NNNGGTCTCCATCCAAAGAC −931 GAAAGGTTGAATGABg10 gDNA was isolated by breaking the cells by vigorous shaking with0.5 mm Zirconia/Silica beads (BioSpec Products, Inc.) in the presence ofphenol-Chloroform, prior to EtOH precipitation and suspension in aTris-EDTA solution (10 mM Tris, 1 mM EDTA). PCR was performed for 35cycles (98° C. for 10 sec, ˜55° C. for 10 sec, and 72° C. for ˜30 sec)with a proof reading polymerase by standard methods and the manufacturesrecommendations (New England BioLabs, NEB). After purification of thePCR amplicons (gel isolation and extraction, DNA Clean and Concentrator,Zymo-Research), the restriction enzyme sites strategically placed in theprimers were then used to cut and ligate each purified PCR amplicon,independently, into pJ-G-A-gal reporter vector (FIG. 17) by ligation asrecommend by the manufacturer (NEB). Standard methods were used toisolate, amplify and purify the each of the resultingpJ-G-A-gal-promoter plasmids. In most cases, the “A” of the reporterstart codon is the +1 position relative to the approximately −1, to−1500 base pair position of the reporter (as indicated in table 3) withthe primers used to amplify the promoters.

In all cases, the sequence of the promoters in these reporter plasmidswas determined using primers 5′ and 3′ of the insertion site in thevector; sequence was obtained across the entire promoter and the cloningjunctions (Genewiz, Inc.), and compared with the respective mRNAsequences to confirm the promoter clone identity. In all cases, thecloning junction between the promoter and the A-gal ORF was designed toposition the A-gal ATG start codon, such that is was in the sameposition as the predicted ATG of the promoters native ORF start codon.

The purified, sequence verified, promoter-reporter constructs werelinearized for transformation and integration into the Pichia genome ofBg10 Pichia cells by electroporation. After restriction enzyme digestionthe DNA was cleaned and concentrated to approximately ˜200 ng/ul beforeeach construct was independently transformed into electro-competentPichia (Bg10, BioGrammatics, Inc.). The Bg10 cells were made competentby incubation of log phase cells with DTT and subsequent sorbitol washes(Pichia Protocols). After electroporation, transformants were selectedon YPD with 800 ug/ml G418 and incubated at 30° C. for ˜3 days. Cellsfrom isolated colonies were patched to similar YPD-G418 plates, andcells from these patches were used to measure the level of expressionfrom the reporter genes.

Expression of the A-gal ORF, as regulated by the different promoters,was determined measuring the A-gal enzymatic activity. Reporter activitywas scored from YPD agar plates made with 100 mM phosphate buffer, pH6.5, and alpha-X-GAL as a chromogenic substrate. Reporter activity wasdetected by a blue colored product in, or around, the cells (FIG. 18).Plates 12 and 23 in FIG. 18, panel A, were replica-plated on differentcarbon sources to plates A and B, respectively, shown in FIG. 18, panelsB and C, respectively. Thus, the designations in FIG. 18, panel A ofplates 12 and 23 refer to FIG. 18, panels B and C, respectively. Thenotations of NO:7, NO:8, etc. refer to SEQ ID NOS (i.e., to the promoterused to express α-galactosidase). The level of α-galactosidaseexpression with the various promoters is noted by 0, +1, +2, +3, +4, and+5 in the eight tables below, with the level of expression increasingfrom 0 to +5. The tables labeled “12A” correspond to the results shownin FIG. 18, panel B for the various carbon sources. The tables labeled“23B” correspond to the results shown in FIG. 18, panel C for thevarious carbon sources.

Relative Alpha Galactosidase Expression, Plate 12/A, Glucose PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Panel/row/column) Sourceexpression (1-5) No: 7 A/A/2 Glucose 3 No: 7 A/A/3 Glucose 3 No: 7 A/A/4Glucose 3 No: 7 A/B/1 Glucose 3 No: 8 A/B/2 Glucose 2 No: 8 A/B/3Glucose 4 No: 8 A/B/4 Glucose 2 No: 8 A/B/5 Glucose 4 Control A/C/1Glucose 0 No: 8 A/C/2 Glucose 2 No: 5 A/C/3 Glucose 3 No: 10 A/C/4Glucose 2 No: 10 A/C/5 Glucose 3 No: 10 A/D/1 Glucose 3 No: 14 A/D/2Glucose 2 No: 14 A/D/3 Glucose 4 No: 14 A/D/4 Glucose 0 No: 14 A/D/5Glucose 5 Control A/E/1 Glucose 0 Control A/E/2 Glucose 0 No: 12 A/E/3Glucose 2 No: 12 A/E/4 Glucose 2 No: 12 A/E/5 Glucose 1 09479 A/F/1Glucose 3 09479 A/F/2 Glucose 2 09479 A/F/3 Glucose 3 No: 13 A/F/4Glucose 4 UPP-513 A/F/5 Glucose 4 UPP-354 A/G/1 Glucose 4 DAS A/G/2Glucose 0 1-0469 A/G/3 Glucose 4 GAP A/G/4 Glucose 3 No: 3 A/H/1 Glucose2 No: 2 A/H/2 Glucose 0 No: 7 A/H/3 Glucose 0 No: 11 A/H/4 Glucose 3 No:11 A/H/5 Glucose 3 No: 3 A/I/1 Glucose 2 No: 3 A/I/2 Glucose 2 No: 3A/I/3 Glucose 1 No: 3 A/I/4 Glucose 2 No: 3 A/J/2 Glucose 1 No: 3 A/J/3Glucose 2

Relative Alpha Galactosidase Expression, Plate 12/A, Glycerol PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Panel/row/column) Sourceexpression (1-5) No: 7 A/A/2 Glycerol 3 No: 7 A/A/3 Glycerol 3 No: 7A/A/4 Glycerol 3 No: 7 A/B/1 Glycerol 3 No: 8 A/B/2 Glycerol 3 No: 8A/B/3 Glycerol 4 No: 8 A/B/4 Glycerol 3 No: 8 A/B/5 Glycerol 4 ControlA/C/1 Glycerol 0 No: 8 A/C/2 Glycerol 2 No: 5 A/C/3 Glycerol 2 No: 10A/C/4 Glycerol 2 No: 10 A/C/5 Glycerol 2 No: 10 A/D/1 Glycerol 2 No: 14A/D/2 Glycerol 2 No: 14 A/D/3 Glycerol 4 No: 14 A/D/4 Glycerol 0 No: 14A/D/5 Glycerol 5 Control A/E/1 Glycerol 0 Control A/E/2 Glycerol 0 No:12 A/E/3 Glycerol 2 No: 12 A/E/4 Glycerol 2 No: 12 A/E/5 Glycerol 109479 A/F/1 Glycerol 3 09479 A/F/2 Glycerol 2 09479 A/F/3 Glycerol 3 No:13 A/F/4 Glycerol 3 UPP-513 A/F/5 Glycerol 3 UPP-354 A/G/1 Glycerol 4DAS A/G/2 Glycerol 0 1-0469 A/G/3 Glycerol 3 GAP A/G/4 Glycerol 2 No: 3A/H/1 Glycerol 3 No: 2 A/H/2 Glycerol 0 No: 2 A/H/3 Glycerol 0 No: 11A/H/4 Glycerol 2 No: 11 A/H/5 Glycerol 3 No: 3 A/I/1 Glycerol 2 No: 3A/I/2 Glycerol 2 No: 3 A/I/3 Glycerol 1 No: 3 A/I/4 Glycerol 2 No: 3A/J/2 Glycerol 1 No: 3 A/J/3 Glycerol 2

Relative Alpha Galactosidase Expression, Plate 12/A, Methanol plates.Promoter FIG. 18 Carbon Relative A-gal (seq ID/name) (Panel/row/column)Source expression (1-5) No: 7 A/A/2 Methanol 2 No: 7 A/A/3 Methanol 3No: 7 A/A/4 Methanol 2 No: 7 A/B/1 Methanol 2 No: 8 A/B/2 Methanol 3 No:8 A/B/3 Methanol 4 No: 8 A/B/4 Methanol 2 No: 8 A/B/5 Methanol 4 ControlA/C/1 Methanol 1 No: 8 A/C/2 Methanol 2 No: 5 A/C/3 Methanol 3 No: 10A/C/4 Methanol 2 No: 10 A/C/5 Methanol 3 No: 10 A/D/1 Methanol 3 No: 14A/D/2 Methanol 2 No: 14 A/D/3 Methanol 5 No: 14 A/D/4 Methanol 0 No: 14A/D/5 Methanol 5 Control A/E/1 Methanol 0 Control A/E/2 Methanol 1 No:12 A/E/3 Methanol 2 No: 12 A/E/4 Methanol 4 No: 12 A/E/5 Methanol 109479 A/F/1 Methanol 4 09479 A/F/2 Methanol 2 09479 A/F/3 Methanol 4 No:13 A/F/4 Methanol 3 UPP-513 A/F/5 Methanol 3 UPP-354 A/G/1 Methanol 4DAS A/G/2 Methanol 2 1-0469 A/G/3 Methanol 3 GAP A/G/4 Methanol 3 No: 3A/H/1 Methanol 2 No: 2 A/H/2 Methanol 2 No: 2 A/H/3 Methanol 2 No: 11A/H/4 Methanol 1 No: 11 A/H/5 Methanol 3 No: 3 A/I/1 Methanol 2 No: 3A/I/2 Methanol 2 No: 3 A/I/3 Methanol 0 No: 3 A/I/4 Methanol 2 No: 3A/J/2 Methanol 1 No: 3 A/J/3 Methanol 2

Relative Alpha Galactosidase Expression, Plate 12-A, Ethanol. PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Panel/row/column) Sourceexpression (1-5) No: 7 A/A/2 Ethanol 2 No: 7 A/A/3 Ethanol 2 No: 7 A/A/4Ethanol 2 No: 7 A/B/1 Ethanol 1 No: 8 A/B/2 Ethanol 0 No: 8 A/B/3Ethanol 3 No: 8 A/B/4 Ethanol 2 No: 8 A/B/5 Ethanol 4 Control A/C/1Ethanol 0 No: 8 A/C/2 Ethanol 1 No: 5 A/C/3 Ethanol 2 No: 10 A/C/4Ethanol 1 No: 10 A/C/5 Ethanol 2 No: 10 A/D/1 Ethanol 2 No: 14 A/D/2Ethanol 1 No: 14 A/D/3 Ethanol 5 No: 14 A/D/4 Ethanol 0 No: 14 A/D/5Ethanol 5 Control A/E/1 Ethanol 0 Control A/E/2 Ethanol 0 No: 12 A/E/3Ethanol 1 No: 12 A/E/4 Ethanol 1 No: 12 A/E/5 Ethanol 0 09479 A/F/1Ethanol 3 09479 A/F/2 Ethanol 2 09479 A/F/3 Ethanol 3 No: 13 A/F/4Ethanol 3 UPP-513 A/F/5 Ethanol 3 UPP-354 A/G/1 Ethanol 4 DAS A/G/2Ethanol 0 1-0469 A/G/3 Ethanol 3 GAP A/G/4 Ethanol 2 No: 3 A/H/1 Ethanol2 No: 2 A/H/2 Ethanol 0 No: 2 A/H/3 Ethanol 1 No: 11 A/H/4 Ethanol 2 No:11 A/H/5 Ethanol 3 No: 3 A/I/1 Ethanol 2 No: 3 A/I/2 Ethanol 1 No: 3A/I/3 Ethanol 0 No: 3 A/I/4 Ethanol 2 No: 3 A/J/2 Ethanol 1 No: 3 A/J/3Ethanol 2

Relative Alpha Galactosidase Expression, Plate 23/B, Glucose PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Plate B/row/column) Sourceexpression (1-5) No: 4 B/A/1 Glucose 0 No: 4 B/A/2 Glucose 0 No: 4 B/A/3Glucose 0 No: 4 B/A/4 Glucose 0 No: 4 B/B/1 Glucose 0 11231 B/B/2Glucose 0 11231 B/B/3 Glucose 0 No: 1 B/B/4 Glucose 0 No: 1 B/B/5Glucose 0 No: 1 B/C/1 Glucose 0 No: 14 B/C/2 Glucose 0 No: 14 B/C/3Glucose 0 No: 14 B/C/4 Glucose 0 No: 14 B/C/5 Glucose 0 No: 14 B/D/1Glucose 2 No: 14 B/D/2 Glucose 1 No: 2 B/D/3 Glucose 0 No: 2 B/D/4Glucose 0 No: 2 B/D/5 Glucose 0 No: 2 B/E/1 Glucose 0 No: 2 B/E/2Glucose 0 No: 2 B/E/3 Glucose 0 No: 2 B/E/4 Glucose 0 No: 2 B/E/5Glucose 0 UPP-513 B/F/1 Glucose 4 UPP-345 B/F/2 Glucose 4 DAS B/F/3Glucose 0 1-0469 B/F/4 Glucose 3 GAP B/F/5 Glucose 4 09476 B/G/1 Glucose2 UPP 222 B/G/2 Glucose 3 No: 13 B/G/3 Glucose 4 No: 13 B/G/4 Glucose 4No: 2 B/H/1 Glucose 0 No: 2 B/H/2 Glucose 0 No: 13 B/H/3 Glucose 4 No: 2B/H/4 Glucose 0 No: 3 B/I/2 Glucose 0 No: 2 B/I/3 Glucose 0 No: 3 B/I/4Glucose 0 No: 3 B/J/3 Glucose 0

Relative Alpha Galactosidase Expression, Plate 23/B, Glycerol PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Plate B/row/column) Sourceexpression (1-5) No: 4 B/A/1 Glycerol 0 No: 4 B/A/2 Glycerol 0 No: 4B/A/3 Glycerol 0 No: 4 B/A/4 Glycerol 0 No: 4 B/B/1 Glycerol 0 11231B/B/2 Glycerol 0 11231 B/B/3 Glycerol 0 No: 1 B/B/4 Glycerol 0 No: 1B/B/5 Glycerol 0 No: 1 B/C/1 Glycerol 0 No: 14 B/C/2 Glycerol 0 No: 14B/C/3 Glycerol 0 No: 14 B/C/4 Glycerol 0 No: 14 B/C/5 Glycerol 0 No: 14B/D/1 Glycerol 2 No: 14 B/D/2 Glycerol 1 No: 2 B/D/3 Glycerol 0 No: 2B/D/4 Glycerol 0 No: 2 B/D/5 Glycerol 0 No: 2 B/E/1 Glycerol 0 No: 2B/E/2 Glycerol 0 No: 2 B/E/3 Glycerol 0 No: 2 B/E/4 Glycerol 0 No: 2B/E/5 Glycerol 0 UPP-513 B/F/1 Glycerol 4 UPP-345 B/F/2 Glycerol 4 DASB/F/3 Glycerol 0 1-0469 B/F/4 Glycerol 3 GAP B/F/5 Glycerol 4 09476B/G/1 Glycerol 2 UPP 222 B/G/2 Glycerol 3 No: 13 B/G/3 Glycerol 4 No: 13B/G/4 Glycerol 4 No: 2 B/H/1 Glycerol 0 No: 2 B/H/2 Glycerol 0 No: 13B/H/3 Glycerol 4 No: 2 B/H/4 Glycerol 0 No: 3 B/I/2 Glycerol 0 No: 2B/I/3 Glycerol 0 No: 3 B/I/4 Glycerol 0 No: 3 B/J/3 Glycerol 0

Relative Alpha Galactosidase Expression, plate 23-B, Methanol PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Plate/row/column) Sourceexpression (1-5) No: 4 B/A/1 Methanol 4 No: 4 B/A/2 Methanol 4 No: 4B/A/3 Methanol 4 No: 4 B/A/4 Methanol 4 No: 4 B/B/1 Methanol 4 11231B/B/2 Methanol 2 11231 B/B/3 Methanol 3 No: 1 B/B/4 Methanol 4 No: 1B/B/5 Methanol 5 No: 1 B/C/1 Methanol 4 No: 14 B/C/2 Methanol 3 No: 14B/C/3 Methanol 2 No: 14 B/C/4 Methanol 1 No: 14 B/C/5 Methanol 0 No: 14B/D/1 Methanol 2 No: 14 B/D/2 Methanol 1 No: 2 B/D/3 Methanol 3 No: 2B/D/4 Methanol 3 No: 2 B/D/5 Methanol 3 No: 2 B/E/1 Methanol 4 No: 2B/E/2 Methanol 2 No: 2 B/E/3 Methanol 3 No: 2 B/E/4 Methanol 3 No: 2B/E/5 Methanol 3 UPP-513 B/F/1 Methanol 5 UPP-345 B/F/2 Methanol 5 DASB/F/3 Methanol 3 1-0469 B/F/4 Methanol 3 GAP B/F/5 Methanol 3 09476B/G/1 Methanol 2 UPP 222 B/G/2 Methanol 4 No: 13 B/G/3 Methanol 4 No: 13B/G/4 Methanol 4 No: 2 B/H/1 Methanol 2 No: 2 B/H/2 Methanol 1 No: 13B/H/3 Methanol 4 No: 2 B/H/4 Methanol 1 No: 3 B/I/2 Methanol 3 No: 2B/I/3 Methanol 2 No: 3 B/I/4 Methanol 3 No: 3 B/J/3 Methanol 3

Relative Alpha Galactosidase Expression, Plate 23-B, Ethanol PromoterFIG. 18 Carbon Relative A-gal (seq ID/name) (Plate B/row/column) Sourceexpression (1-5) No: 4 B/A/1 Ethanol 0 No: 4 B/A/2 Ethanol 0 No: 4 B/A/3Ethanol 0 No: 4 B/A/4 Ethanol 0 No: 4 B/B/1 Ethanol 0 11231 B/B/2Ethanol 0 11231 B/B/3 Ethanol 0 No: 1 B/B/4 Ethanol 0 No: 1 B/B/5Ethanol 0 No: 1 B/C/1 Ethanol 0 No: 14 B/C/2 Ethanol 0 No: 14 B/C/3Ethanol 0 No: 14 B/C/4 Ethanol 0 No: 14 B/C/5 Ethanol 0 No: 14 B/D/1Ethanol 1 No: 14 B/D/2 Ethanol 0 No: 2 B/D/3 Ethanol 0 No: 2 B/D/4Ethanol 0 No: 2 B/D/5 Ethanol 0 No: 2 B/E/1 Ethanol 0 No: 2 B/E/2Ethanol 0 No: 2 B/E/3 Ethanol 0 No: 2 B/E/4 Ethanol 0 No: 2 B/E/5Ethanol 0 UPP-513 B/F/1 Ethanol 4 UPP-345 B/F/2 Ethanol 4 DAS B/F/3Ethanol 0 1-0469 B/F/4 Ethanol 3 GAP B/F/5 Ethanol 4 09476 B/G/1 Ethanol2 UPP 222 B/G/2 Ethanol 3 No: 13 B/G/3 Ethanol 4 No: 13 B/G/4 Ethanol 4No: 2 B/H/1 Ethanol 0 No: 2 B/H/2 Ethanol 0 No: 13 B/H/3 Ethanol 4 No: 2B/H/4 Ethanol 0 No: 3 B/I/2 Ethanol 0 No: 2 B/I/3 Ethanol 0 No: 3 B/I/4Ethanol 0 No: 3 B/J/3 Ethanol 0

1. (canceled) 2-76. (canceled)
 77. A method of producing a protein, themethod comprising the step of culturing in a culture medium a host cellcomprising a first expression cassette comprising an isolated nucleicacid at least 95% identical to a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:6, or at least 95% identical to a fragment thereof, operably linked to aheterologous coding sequence encoding a protein, wherein the isolatednucleic acid comprises the sequence of a methanol inducible Pichiapastoris promoter, wherein the fragment extends about 300 nucleotides,about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, orabout 700 nucleotides upstream from the 3′ end of SEQ ID NO:1, SEQ IDNO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, wherein the fragment is acontinuous fragment of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO: 6, wherein the fragment comprises a TATA box sequence to directinitiation of transcription, wherein the culturing is done underconditions permitting expression of the protein, and wherein the proteinis expressed using the first expression cassette in combination with asecond expression cassette, said second cassette comprising (a) theheterologous coding sequence encoding the protein operably linked to anisolated nucleic acid having a sequence comprising SEQ ID NO: 2 or SEQID NO: 4, wherein SEQ ID NO: 2 or SEQ ID NO: 4 have promoter activity;or (b) the heterologous coding sequence encoding the protein operablylinked to an isolated nucleic acid having a sequence comprising SEQ IDNO: 15 or SEQ ID NO: 16, wherein SEQ ID NO: 15 or SEQ ID NO: 16 havepromoter activity; or (c) the heterologous coding sequence encoding theprotein operably linked to a different AOX promoter than SEQ ID NO:15 orSEQ ID NO:16, an FLD promoter, a DAS promoter, or a methanol-regulatedpromoter; or (d) the isolated nucleic acid at least 95% identical to anucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, and SEQ ID NO: 6, or at least 95% identical to afragment thereof, operably linked to the heterologous coding sequenceencoding the protein.
 78. The method of claim 77 wherein the isolatednucleic acid in the first expression cassette is at least 98% identicalto a nucleic acid selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6, or at least 98% identicalto the fragment thereof.
 79. The method of claim 77 wherein the sequenceof the isolated nucleic acid in the first expression cassette is anucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, and SEQ ID NO: 6, or the fragment thereof.
 80. Themethod of claim 77 wherein the sequence of the isolated nucleic acid inthe first expression cassette is a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:6.
 81. The method of claim 77 wherein the heterologous coding sequenceencodes a protein selected from the group consisting of a toxin, anantibody, a hormone, an enzyme, a growth factor, a cytokine, astructural protein, an immunogenic protein, and a cell signalingprotein.
 82. The method of claim 77 wherein the heterologous codingsequence encodes an enzyme for use in animal feed.
 83. The method ofclaim 82 wherein the enzyme is selected from the group consisting of amannanase, an amylase, a glucanase, a protease, a cellulase, and axylanase.
 84. The method of claim 82 wherein the enzyme is a phytase.85. The method of claim 82 wherein the enzyme is a galactosidase.
 86. Ahost cell comprising a first expression cassette comprising an isolatednucleic acid at least 95% identical to a nucleic acid selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ IDNO: 6, or at least 95% identical to a fragment thereof, operably linkedto a heterologous coding sequence encoding a protein, wherein theisolated nucleic acid comprises the sequence of a methanol induciblePichia pastoris promoter, wherein the fragment extends about 300nucleotides, about 400 nucleotides, about 500 nucleotides, about 600nucleotides, or about 700 nucleotides upstream from the 3′ end of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, wherein the fragmentis a continuous fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, orSEQ ID NO: 6, wherein the fragment comprises a TATA box sequence todirect initiation of transcription, and a second expression cassette,said second cassette comprising (a) the heterologous coding sequenceencoding the protein operably linked to an isolated nucleic acid havinga sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4, wherein SEQ ID NO: 2or SEQ ID NO: 4 have promoter activity; or (b) the heterologous codingsequence encoding the protein operably linked to an isolated nucleicacid having a sequence comprising SEQ ID NO: 15 or SEQ ID NO: 16,wherein SEQ ID NO: 15 or SEQ ID NO: 16 have promoter activity; or (c)the heterologous coding sequence encoding the protein operably linked toa different AOX promoter than SEQ ID NO:15 or SEQ ID NO:16, an FLDpromoter, a DAS promoter, or a methanol-regulated promoter; or (d) theisolated nucleic acid at least 95% identical to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, or SEQ ID NO: 6, or at least 95% identical to afragment thereof, operably linked to the heterologous coding sequenceencoding the protein.
 87. The host cell of claim 86 wherein the isolatednucleic acid in the first expression cassette is at least 98% identicalto SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, or atleast 98% identical to the fragment thereof.
 88. The host cell of claim86 wherein the sequence of the isolated nucleic acid in the firstexpression cassette is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO: 6, or the fragment thereof.
 89. The host cell of claim 86 whereinthe sequence of the isolated nucleic acid in the first expressioncassette is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6.90. The host cell of claim 86 wherein the heterologous coding sequenceencodes a protein selected from the group consisting of a toxin, anantibody, a hormone, an enzyme, a growth factor, a cytokine, astructural protein, an immunogenic protein, and a cell signalingprotein.
 91. The host cell of claim 86 wherein the heterologous codingsequence encodes an enzyme for use in animal feed.
 92. The host cell ofclaim 91 wherein the enzyme is selected from the group consisting of amannanase, an amylase, a glucanase, a protease, a cellulase, and axylanase.
 93. The host cell of claim 91 wherein the enzyme is a phytase.94. The host cell of claim 91 wherein the enzyme is a galactosidase.